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1995 - 19982

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Subject: search.filters.subject.Haemproteins

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    X-ray crystallographic analysis of cytochrome c' from two bacterial species : this thesis is submitted as partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University
    (Massey University, 1995) Dobbs, Aaron John; Dobbs, Aaron John
    The structure of cytochrome c' from two closely related bacterial species, Alcaligenes sp and Alcaligenes denitrificans, have been determined from X-ray diffraction data to 1.80 Å and 2.15 Å resolution respectively using the anomalous scattering of the single iron atom in each to identify and refine a weak molecular replacement solution. Molecular replacement studies, with the program AMORE, used two isomorphous data sets (from the two species), two independent search models (the cytochromes c' from Rhodospirillum molischianum and Rhodospirillum rubrum), both with and without sidechains, and two different resolution ranges (10.0-4.0 Å and 15.0-3.5 Å) to generate a large number of potential solutions. No single solution stood out and none appeared consistently. The iron position in each structure was then determined from its anomalous scattering contribution and all molecular replacement solutions were discarded which did not (i) place the iron correctly and (ii) orient the molecule such that a crystallographic 2-fold axis generated a dimer like those of the two search models. Finally, electron density maps phased solely by the iron anomalous scattering were calculated. As these were combined and subjected to solvent flattening and histogram matching (program SQUASH), correlation with the remaining molecular replacement solutions identified one as correct and enabled it to be improved and subjected to preliminary refinement. The correctness of the solution was confirmed by parallel isomorphous replacement studies. Both crystal structures have been refined using least-squares methods, to give final R-factors of 0.184 for the Alcaligenes sp cytochrome c' (953 protein atoms and 89 solvent molecules) and 0.167 for the Alcaligenes denitrificans cytochrome c' (950 protein atoms and 75 solvent molecules). Analysis and comparisons of these structures with three cytochrome c' structures previously determined show that despite the low level of sequence conservation among the family members the gross overall structure is maintained (four-α-helix bundle). The configuration of the iron ligands and of the surrounding haem environment are very similar indicating that although residues are not conserved they are replaced by residues which fulfil a similar function. There are relatively few sidechain...sidechain interactions in cytochromes c' which probably reflects the fact that the majority of stabilising i interactions are hydrophobic in nature. This hydrophobic packing could also explain the lack of sequence identity between the different species of cytochrome c' as strict conservation of the residues is not required. All the structures do have a similar haem stabilisation framework, with an approximately ten residue piece of structure situated in the BC loop involved in both haem stabilisation and interhelix stabilisation in all of the cytochromes c' determined. The overall four-helix structure is also discussed in terms of its parameters with respect to the current interest in the de novo design of proteins.
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    X-ray crystallographic analyses of the structures of two heme proteins : a thesis submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in the Institute of Molecular BioSciences at Massey University, New Zealand
    (Massey University, 1998) Sutherland-Smith, Andrew John
    During human development three embryonic hemoglobins are synthesised prior to formation of the placenta. These hemoglobins function to scavenge oxygen from the mother's interstitial fluid enabling embryonic respiration. The human Gower II embryonic haemoglobin (α2ε2) has been crystallized in its carbonmonoxy form, and its structure determined by X-ray crystallography. The structure was solved by molecular replacement and refined at 2.9 Å. The Gower II hemoglobin tetramer is intermediate between the adult hemoglobin R and R2 states, though closer to R2. The tertiary structure of the α subunit is essentially identical when compared to that found in the adult (α2β2) and fetal (α2γ2) hemoglobins. The embryonic ε subunit has a very similar structure to the homologous adult β and fetal γ subunits, although with small differences at the N-terminus and in the A helix. Amino acid substitutions can be identified that may play a role in the altered response of the Gower II haemoglobin to allosteric effectors, in particular chloride ions. Nitrite reductase from Pseudomonas stutzeri is a periplasmic heme enzyme responsible for the reduction of nitrite to nitric oxide. This reaction is the second step in the bacterial denitrification pathway, during which nitrate acts as the terminal electron acceptor for anaerobic respiration and is consequently reduced to nitrogen gas. Nitrite reductase from Pseudomonas stutzeri JM300 has been crystallized in the oxidised state and X-ray diffraction data collected to a resolution of 2.8 Å. The structure has been solved by the method of molecular replacement. The structure of the enzyme is dimeric, with each monomer comprised of two domains. The smaller N-terminal domain covalently binds a c heme group within an all α-helical fold similar to that of the class l c-type cytochromes. The larger C-terminal domain consists of an eight-bladed β-propeller structure that coordinates a d1 heme, a cofactor unique to this class of enzyme. The relative positions of the two domains, and hence the orientations of the bound heme groups are markedly different compared to homologous enzymes from other species.